6. Project Summary/Abstract Cripto is a GPI-anchored developmental oncoprotein that is selectively overexpressed in a wide range of human tumors including ~80% of human breast cancers. Cripto promotes the tumor phenotype via its dual effects on activin/Nodal/TGF-[unreadable] and MAPK/PI3K signaling pathways. Activin, Nodal and TGF-[unreadable] are structurally related TGF-[unreadable] superfamily members that activate the Smad2/3 signaling pathway and control proliferation, differentiation and morphogenesis in normal developing and adult tissues and in disease states such as cancer. Cripto modulates activin/Nodal/TGF-[unreadable] signaling by forming complexes with these ligands and their respective signaling receptors and has been shown to play essential physiological roles as an obligatory Nodal co-receptor in stem cells and during embryogenesis. By contrast, we have demonstrated that Cripto antagonizes activin and TGF-[unreadable] signaling and inhibits the antiproliferative effects of these ligands on mammary epithelial and prostate carcinoma cell lines. In addition to its effects on TGF-[unreadable] ligand signaling, soluble forms of Cripto have tumor growth factor activity and activate ras/raf/MAPK and PI3K/Akt pathways via an unidentified cell surface receptor. In an effort to identify this receptor, we conducted a protein-protein interaction screen using Cripto as bait. This screen led to the identification of GRP78, an ER chaperone that is selectively expressed at the surface of tumor cells where it has receptor function that promotes proliferation and survival. We have shown that GRP78 and Cripto form a cell surface complex and collaborate to promote Cripto effects on TGF-[unreadable] signaling. Our preliminary results further indicate that soluble Cripto binding to cell surface GRP78 causes Akt phosphorylation and increased cellular proliferation. Based on these findings, we hypothesize that GRP78 functions as a cell surface Cripto receptor/co-factor that mediates oncogenic effects of Cripto on activin/Nodal/TGF-[unreadable] and MAPK/PI3K signaling pathways. Under Aim I of this proposal we will identify the molecular determinants on Cripto and GRP78 that are required for their specific binding interaction and we will also elucidate the signaling mechanisms of the Cripto/GRP78 complex. We also propose to develop reagents designed to bind Cripto or GRP78 and thereby disrupt Cripto/GRP78 complex formation and signaling. Aim II proposes to test the hypothesis that cell surface GRP78 mediates oncogenic Cripto effects on cellular proliferation, migration, invasion, angiogenesis and morphogenesis in vitro. Experiments outlined in Aim II are also designed to characterize the contribution of the Cripto/GRP78 interaction to tumor growth in vivo. We will inject human tumor cells with manipulated levels of Cripto and/or GRP78 into nude mice and then monitor tumor growth. In addition, we will use somatic gene transfer to introduce Cripto and/or GRP78 directly into mammary epithelial cells in normal adult mice and then characterize the ability of these proteins to initiate tumor growth. Together, these studies will illuminate novel therapeutic targets and reagents for the management of neoplastic disease.